Transcriptional co-repressor that interacts with nuclear hormone receptors and uses therefor

ABSTRACT

In accordance with the present invention, there are provide novel receptor interacting factors, referred to herein as “SMRT”, i.e., a silencing mediator (co-repressor) for retinoic acid receptor (RAR) and thyroid hormone receptor (TR). SMRT is a novel protein whose association with RAR and TR both in solution and on DNA response elements is destabilized by ligand. The interaction of SMRT with mutant receptors correlates with the transcriptional silencing activities of receptors. In vivo, SMRT functions as a potent co-repressor. A GAL4 DNA binding domain (DBD) fusion of SMRT behaves as a frank repressor of a GAL4-dependent reporter. Together, these data identify a novel class of cofactor which is believed to represent an important mediator of hormone action.

RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. patentapplication Ser. No. 09/337,384, filed Jun. 21, 1999, currently pending,which is in turn a divisional application of U.S. patent applicationSer. No. 08/522,726, filed on Sep. 1, 1995, now issued as U.S. Pat. No.6,489,441, the entire contents of each of which are hereby incorporatedby reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates to intracellular receptors, methodsfor the modulation thereof, and methods for the identification of novelligands therefor. In a particular aspect, the present invention relatesto methods for the identification of compounds which function as ligands(or ligand precursors) for intracellular receptors. In another aspect,the present invention relates to novel chimeric constructs and usestherefor.

BACKGROUND OF THE INVENTION

[0003] A central problem in eukaryotic molecular biology continues to bethe elucidation of molecules and mechanisms that mediate specific generegulation. As part of the scientific attack on this problem, a greatdeal of work has been done in efforts to identify ligands (i.e.,exogenous inducers) which are capable of mediating specific generegulation. Additional work has been done in efforts to identify othermolecules involved in specific gene regulation.

[0004] Although much remains to be learned about the specifics of generegulation, it is known that ligands modulate gene transcription byacting in concert with intracellular components, including intracellularreceptors and discrete DNA sequences known as hormone response elements(HREs).

[0005] The identification of compounds which directly or indirectlyinteract with intracellular receptors, and thereby affect transcriptionof hormone-responsive genes, would be of significant value, e.g., fortherapeutic applications.

[0006] Transcriptional silencing mediated by nuclear receptors plays animportant role in development, cell differentiation, and is directlylinked to the oncogenic activity of v-erbA. The mechanism underlyingthis effect is unknown but is one key to understanding the molecularbasis of hormone action. Accordingly, the identification of componentsinvolved in transcriptional silencing would represent a great advance incurrent understanding of mechanisms that mediate specific generegulation.

[0007] Other information helpful in the understanding and practice ofthe present invention can be found in commonly assigned U.S. Pat. Nos.5,071,773 and 4,981,784; and U.S. patent application Ser. No. 325,240,filed Mar. 17, 1989 (now issued as U.S. Pat. No. 5,597,693, based oncontinuation in part application Ser. No. 494,618, filed Mar. 16, 1990);Ser. No. 370,407, filed Jun. 22, 1989 (now issued as U.S. Pat. No.5,260,432) and Ser. No. 438,757, filed Nov. 16, 1989 (now issued as U.S.Pat. No. 5,091,518), all of which are hereby incorporated herein byreference in their entirety.

BRIEF DESCRIPTION OF THE INVENTION

[0008] In accordance with the present invention, we have discovered anovel receptor interacting factor, referred to herein as “SMRT”, i.e., asilencing mediator (co-repressor) for retinoic acid receptor (RAR) andthyroid hormone receptor (TR). SMRT is a novel protein whose associationwith RAR and TR both in solution and on DNA response elements isdestabilized by ligand. The interaction of SMRT with mutant receptorscorrelates with the transcriptional silencing activities of receptors.

[0009] In vivo, SMRT functions as a potent co-repressor. A GAL4 DNAbinding domain (DBD) fusion of SMRT behaves as a frank repressor of aGAL4-dependent reporter. Together, these data identify a novel class ofcofactor which is believed to represent an important mediator of hormoneaction.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1 shows the quantitation by phosphoimager of a dose-dependentdissociation of SMRT from RAR or TR by all-trans retinoic acid (atRA) orthyroid hormone (triiodothyronine or T3).

[0011]FIG. 2 presents amino acid (aa) sequences of SMRT (Genbankaccession number U37146; SEQ ID NO:1). The aa sequence presented inparentheses (i.e., residues 1330-1376) is an alternatively splicedinsert which is not present in the original two-hybrid clone (C-SMRT, aa981 to C-terminal end). The proline-rich N-terminal domain (aa 1-160)and the glutamine-rich region (aa 1061-1132), as well as the ERDR-richand SG-rich regions (underlined), are also indicated. The C-terminalregion of SMRT (aa 1201 to C-terminal end) shows 48% aa identity toRIP13 (Seol et al., Molecular Endocrinology 9:72-85 (1995)). The rest ofthe sequence of RIP13 shows 22% aa identity to SMRT (aa 819-1200)

[0012]FIG. 3 collectively illustrates mediation of the silencing effectof hRARCα and hTRβ by SMRT in vivo.

[0013]FIG. 3(A) illustrates that v-erbA reverses the silencing effect ofGAL-RAR (GAL4 DBD-hRARα 156-462) while SMRT restores the silencingeffect.

[0014]FIG. 3(B) illustrates that the RAR403 truncation mutant reversesthe silencing effect of GAL-TR (GAL4 DBD-hTRβ 173-456) while SMRTrestores the silencing effect.

[0015]FIG. 3(C) illustrates that v-erbA and full length SMRT or C-SMRThave no effect on GAL-VP16 activity.

[0016]FIG. 3(D) illustrates that a GAL4 DBD fusion of full length SMRTsuppresses the thymidine kinase basal promoter activity containing fourGAL4 binding sites. The fold of repression was calculated by dividingthe normalized luciferase activity transfected with the GAL4 DBD aloneby those transfected with indicated amount of GAL DBD fusion constructs.

DETAILED DESCRIPTION OF THE INVENTION

[0017] In accordance with the present invention, there are providedco-suppressors of steroid/thyroid hormone receptor activity, saidco-suppressors having a structure and function characteristic of thesilencing mediator for retinoic acid receptor and thyroid hormonereceptor.

[0018] Co-suppressors contemplated by the present invention havesubstantially the same sequence as residues 1-1329 plus 1376-1495, asset forth in SEQ ID NO:1, optionally further comprising the amino acidresidues set forth in SEQ ID NO:2 (i.e., residues 1330-1375 of SEQ IDNO:1).

[0019] The phrase “substantially the same” is used herein in referenceto the nucleotide sequence of DNA, the ribonucleotide sequence of RNA,or the amino acid sequence of protein, that have slight andnon-consequential sequence variations from the actual sequencesdisclosed herein. Species that are substantially the same are consideredto be equivalent to the disclosed sequences and as such are within thescope of the appended claims. In this regard, “slight andnon-consequential sequence variations” mean that sequences that aresubstantially the same as the DNA, RNA, or proteins disclosed andclaimed herein are functionally equivalent to the sequences disclosedand claimed herein. Functionally equivalent sequences will function insubstantially the same manner to produce substantially the samecompositions as the nucleic acid and amino acid compositions disclosedand claimed herein. In particular, functionally equivalent DNAs encodeproteins that are the same as those disclosed herein or that haveconservative amino acid variations, such as substitution of a non-polarresidue for another non-polar residue or a charged residue for asimilarly charged residue. These changes include those recognized bythose of skill in the art as those that do not substantially alter thetertiary structure of the protein.

[0020] In accordance with another aspect of the present invention, thereare provided antibodies raised against the above-describedco-suppressor. Such antibodies can be employed for studying tissuelocalization of invention co-repressor, the structure of functionaldomains, the purification of receptors, as well as in diagnosticapplications, therapeutic applications, and the like. Preferably, fortherapeutic applications, the antibodies employed will be monoclonalantibodies.

[0021] The above-described antibodies can be prepared employing standardtechniques, as are well known to those of skill in the art, using theinvention co-repressor or portions thereof as antigens for antibodyproduction. Both anti-peptide and anti-fusion protein antibodies can beused [see, for example, Bahouth et al. (1991) Trends Pharmacol Sci. vol.12:338-343; Current Protocols in Molecular Biology (Ausubel et al.,eds.) John Wiley and Sons, New York (1989)]. Factors to consider inselecting portions of invention co-repressor for use as immunogen (aseither a synthetic peptide or a recombinantly produced bacterial fusionprotein) include antigenicity, accessibility (i.e., where the selectedportion is derived from, e.g., the ligand binding domain, DNA bindingdomain, dimerization domain, and the like), uniqueness of the particularportion selected (relative to known receptors and co-suppressorstherefor), and the like.

[0022] In accordance with yet another aspect of the present invention,there are provided methods to block the repressing effect of inventionco-suppressors, said method comprising administering an effective amountof an antibody as described herein. Alternatively, a silencing domain ofa nuclear receptor can be employed. Those of skill in the art canreadily determine suitable methods for administering said antibodies,and suitable quantities for administration, which will vary depending onnumerous factors, such as the indication being treated, the condition ofthe subject, and the like.

[0023] In accordance with a still further aspect of the invention, thereare provided isolated polynucleic acids encoding the above-describedco-suppressor. In addition, there are also provided vectors containingthe above-described polynucleic acid.

[0024] In accordance with a still further aspect of the presentinvention, there are provided complexes comprising the above-describedco-suppressor and a homodimeric or heterodimeric member of thesteroid/thyroid hormone superfamily of receptors, wherein said membercontains a silencing domain which represses basal level promoteractivity of target genes. Homodimeric or heterodimeric members of thesteroid/thyroid hormone superfamily of receptors contemplated for useherein include thyroid hormone receptor homodimer, thyroid hormonereceptor-retinoid X receptor heterodimer, retinoic acid receptorhomodimer, retinoic acid receptor-retinoid X receptor heterodimer,retinoid X receptor homodimer, and the like.

[0025] The above-described complexes optionally further comprise aresponse element for the member of the steroid/thyroid hormonesuperfamily of receptors. Such response elements are well known in theart. Thus, for example, RAR response elements are composed of at leastone direct repeat of two or more half sites separated by a spacer offive nucleotides. The spacer nucleotides can independently be selectedfrom any one of A, C, G or T. Each half site of response elementscontemplated for use in the practice of the invention comprises thesequence

-RGBNNM-,

[0026] wherein

[0027] R is selected from A or G;

[0028] B is selected from G, C, or T;

[0029] each N is independently selected from A, T, C, or G; and

[0030] M is selected from A or C;

[0031] with the proviso that at least 4 nucleotides of said -RGBNNM-sequence are identical with the nucleotides at corresponding positionsof the sequence -AGGTCA-. Response elements employed in the practice ofthe present invention can optionally be preceded by N_(x) wherein xfalls in the range of 0 up to 5.

[0032] Similarly, TR response elements can be composed of the same halfsite repeats, with a spacer of four nucleotides. Alternatively,palindromic constructs as have been described in the art are alsofunctional as TR response elements.

[0033] The above-described co-repressor/dimeric receptor complexes canbe dissociated by contacting complex with a ligand for the member of thesteroid/thyroid hormone superfamily of receptors.

[0034] As employed herein, the term “ligand (or ligand precursor) for amember of the steroid/thyroid hormone superfamily of receptors” (i.e.,intracellular receptor) refers to a substance or compound which, in itsunmodified form (or after conversion to its “active” form), inside acell, binds to receptor protein, thereby creating a ligand/receptorcomplex, which in turn can activate an appropriate hormone responseelement. A ligand therefore is a compound which acts to modulate genetranscription for a gene maintained under the control of a hormoneresponse element, and includes compounds such as hormones, growthsubstances, non-hormone compounds that modulate growth, and the like.Ligands include steroid or steroid-like hormone, retinoids, thyroidhormones, pharmaceutically active compounds, and the like. Individualligands may have the ability to bind to multiple receptors.

[0035] Accordingly, as employed herein, “putative ligand” (also referredto as “test compound”) refers to compounds such as steroid orsteroid-like hormones, pharmaceutically active compounds, and the like,which are suspected to have the ability to bind to the receptor ofinterest, and to modulate transcription of genes maintained under thecontrol of response elements recognized by such receptor.

[0036] Examples of known ligands include all-trans-retinoic acid (ligandfor retinoic acid receptor), 9-cis-retinoic acid (ligand for retinoid Xreceptor), thyroid hormone (ligand for thyroid hormone receptor),1,25-dihydroxy vitamin D₃ (ligand for vitamin D₃ receptor), and thelike.

[0037] In accordance with another aspect of the present invention, thereis provided a method to repress the activity of a member of thesteroid/thyroid hormone superfamily of receptors containing a silencingdomain which represses basal level promoter activity of target genes,said method comprising contacting said member of the steroid/thyroidhormone superfamily of receptors with a sufficient quantity of aco-suppressor as described hereinabove so as to repress the activity ofsaid member. Members of the superfamily contemplated for repression inaccordance with this aspect of the present invention include thyroidhormone receptor, retinoic acid receptor, vitamin D receptor, peroxisomeproliferator activated receptor, and the like.

[0038] In accordance with yet another aspect of the present invention,there is provided a method to identify compounds which relieve thesuppression of steroid/thyroid hormone receptor activity caused by aco-suppressor as described hereinabove, said method comprising comparingthe size of the above-described co-suppressor/dimeric receptor complex(i.e., complexes comprising the above-described co-suppressor and ahomodimeric or heterodimeric member of the steroid/thyroid hormonesuperfamily of receptors) upon exposure to test compound, relative tothe size of said complex in the absence of test compound. An observedsize corresponding to intact complex is indicative of an inactivecompound, while an observed size that reflects dissociation of thecomplex is indicative of a compound that disrupts the complex, therebyrelieving the suppression caused thereby. Optionally, the complexemployed in this assay further comprises a response element for saidmember of the steroid/thyroid hormone superfamily of receptors.

[0039] The size of the above-described complex can readily be determinedemploying various techniques available in the art. For example,electrophoretic mobility shift assays (EMSA) can be employed (whereinreceptor alone or receptor-co-suppressor complex is bound to target DNAand the relative mobility thereof determined). Those of skill in the artcan readily identify other methodology which can be employed todetermine the size of the complex as a result of exposure to putativeligand.

[0040] In accordance with a still further aspect of the presentinvention, there is provided a method to identify compounds whichrelieve the suppression of steroid/thyroid hormone receptor activitycaused by a co-suppressor as described hereinabove, withoutsubstantially activating said receptor, said method comprising:

[0041] comparing the reporter signal produced by two differentexpression systems in the absence and presence of test compound,

[0042] wherein said first expression system comprises a complexcomprising:

[0043] a homodimeric or heterodimeric member of the steroid/thyroidhormone superfamily of receptors selected from thyroid hormone receptorhomodimer, thyroid hormone receptor-retinoid X receptor heterodimer,retinoic acid receptor homodimer, or retinoic acid receptor-retinoid Xreceptor heterodimer,

[0044] a response element for said member of the steroid/thyroid hormonesuperfamily of receptors, wherein said response element is operativelylinked to a reporter, and

[0045] optionally, invention co-suppressor, and

[0046] wherein said second expression system comprises a complexcomprising:

[0047] a homodimeric or heterodimeric form of the same member of thesteroid/thyroid hormone superfamily of receptors as employed in saidfirst expression system, wherein said member is mutated such that itretains hormone dependent activation activity but has lost its abilityto repress basal level promoter activity of target genes,

[0048] the same response element-reporter combination as employed insaid first expression system, and

[0049] optionally, invention co-suppressor, and thereafter

[0050] selecting those compounds which provide:

[0051] a higher reporter signal upon exposure of said compound to saidfirst expression system, relative to reporter signal in the absence ofsaid compound, and

[0052] substantially the same reporter signal upon exposure of saidcompound to said second expression system, relative to reporter signalin the absence of said compound,

[0053] wherein said selected compounds are capable of relieving thesuppression of steroid/thyroid hormone receptor activity caused by aco-suppressor having a structure and function characteristic of thesilencing mediator for retinoic acid and thyroid receptors, butsubstantially lacking the ability to activate steroid/thyroid hormonereceptor activity.

[0054] The addition of invention co-suppressor is optional in theabove-described assay because it is present endogenously in most hostcells employed for such assays. It is preferred, to ensure the presenceof a fairly constant amount of co-suppressor, and to ensure thatco-suppressor is not a limiting reagent, that co-suppressor be suppliedexogenously to the above-described assays.

[0055] Mutant receptors contemplated for use in the practice of thepresent invention are conveniently produced by expression plasmids,introduced into the host cell by transfection. Mutant receptorscontemplated for use herein include RAR403 homodimers, RAR403-containingheterodimers, TR160 homodimers, TR160-containing heterodimers, and thelike.

[0056] Reporter constructs contemplated for use in the practice of thepresent invention comprise:

[0057] (a) a promoter that is operable in the host cell,

[0058] (b) a hormone response element, and

[0059] (c) a DNA segment encoding a reporter protein,

[0060] wherein the reporter protein-encoding DNA segment is operativelylinked to the promoter for transcription of the DNA segment, and

[0061] wherein the hormone response element is operatively linked to thepromoter for activation thereof.

[0062] Hormone response elements contemplated for use in the practice ofthe present invention are well known in the art, as has been notedpreviously.

[0063] Exemplary reporter genes include chloramphenicol transferase(CAT), luciferase (LUC), beta-galactosidase (β-gal), and the like.Exemplary promoters include the simian virus (SV) promoter or modifiedform thereof (e.g., ΔSV), the thymidine kinase (TK) promoter, themammary tumor virus (MTV) promoter or modified form thereof (e.g.,ΔMTV), and the like [see, for example, Mangelsdorf et al., in Nature345:224-229 (1990), Mangelsdorf et al., in Cell 66:555-561 (1991), andBerger et al., in J. Steroid Biochem. Molec. Biol. 41:733-738 (1992)].

[0064] As used herein in the phrase “operative response elementfunctionally linked to an operative reporter gene”, the word “operative”means that the respective DNA sequences (represented by the terms “GAL4response element” and “reporter gene”) are operational, i.e., work fortheir intended purposes; the word “functionally” means that after thetwo segments are linked, upon appropriate activation by aligand-receptor complex, the reporter gene will be expressed as theresult of the fact that the “GAL4 response element” was “turned on” orotherwise activated.

[0065] In practicing the above-described functional bioassay, theexpression plasmid and the reporter plasmid are co-transfected intosuitable host cells. The transfected host cells are then cultured in thepresence and absence of a test compound to determine if the testcompound is able to produce activation of the promoter operativelylinked to the response element of the reporter plasmid. Thereafter, thetransfected and cultured host cells are monitored for induction (i.e.,the presence) of the product of the reporter gene sequence.

[0066] Any cell line can be used as a suitable “host” for the functionalbioassay contemplated for use in the practice of the present invention.Thus, cells contemplated for use in the practice of the presentinvention include transformed cells, non-transformed cells, neoplasticcells, primary cultures of different cell types, and the like. Exemplarycells which can be employed in the practice of the present inventioninclude Schneider cells, CV-l cells, HuTu80 cells, F9 cells, NTERA2cells, NB4 cells, HL-60 cells, 293 cells, Hela cells, yeast cells, andthe like. Preferred host cells for use in the functional bioassay systemare COS cells and CV-1 cells. COS-l (referred to as COS) cells aremonkey kidney cells that express SV40 T antigen (Tag); while CV-1 cellsdo not express SV40 Tag. The presence of Tag in the COS-1 derivativelines allows the introduced expression plasmid to replicate and providesa relative increase in the amount of receptor produced during the assayperiod. CV-1 cells are presently preferred because they are particularlyconvenient for gene transfer studies and provide a sensitive andwell-described host cell system.

[0067] The above-described cells (or fractions thereof) are maintainedunder physiological conditions when contacted with physiologicallyactive compound. “Physiological conditions” are readily understood bythose of skill in the art to comprise an isotonic, aqueous nutrientmedium at a temperature of about 37° C.

[0068] In accordance with yet another aspect of the present invention,there is provided a method to identify compounds which activatesteroid/thyroid hormone receptor activity, but substantially lack theability to relieve the suppression caused by a co-suppressor asdescribed hereinabove, said method comprising:

[0069] comparing the reporter signal produced by two differentexpression systems in the absence and presence of test compound,

[0070] wherein said first expression system comprises a complexcomprising:

[0071] a homodimeric or heterodimeric member of the steroid/thyroidhormone superfamily of receptors selected from thyroid hormone receptorhomodimer, thyroid hormone receptor-retinoid X receptor heterodimer,retinoic acid receptor homodimer, or retinoic acid receptor-retinoid Xreceptor heterodimer,

[0072] a response element for said member of the steroid/thyroid hormonesuperfamily of receptors, wherein said response element is operativelylinked to a reporter, and

[0073] optionally, invention co-suppressor, and

[0074] wherein said second expression system comprises a complexcomprising:

[0075] a homodimeric or heterodimeric form of the same member of thesteroid/thyroid hormone superfamily of receptors as employed in saidfirst expression system, wherein said member is mutated such that itretains hormone dependent activation activity but has lost its abilityto repress basal level promoter activity of target genes,

[0076] the same response element-reporter combination as employed insaid first expression system, and

[0077] optionally, invention co-suppressor, and thereafter

[0078] selecting those compounds which provide:

[0079] a higher reporter signal upon exposure of said compound to saidsecond expression system, relative to reporter signal in the absence ofcompound, and

[0080] substantially the same reporter signal upon exposure of saidcompound to said first expression system, relative to reporter signal inthe absence of said compound,

[0081] wherein said selected compounds are capable of activatingsteroid/thyroid hormone receptor activity, but substantially lacking theability to relieve the suppression caused by a co-suppressor having astructure and function characteristic of the silencing mediator forretinoic acid and thyroid receptors.

[0082] In accordance with a still further aspect of the presentinvention, there is provided a method to identify compounds whichrelieve the suppression of steroid/thyroid hormone receptor activitycaused by a co-suppressor as described hereinabove, and activate saidreceptor, said method comprising:

[0083] comparing the reporter signal produced by two differentexpression systems in the absence and presence of test compound,

[0084] wherein said first expression system comprises a complexcomprising:

[0085] a homodimeric or heterodimeric member of the steroid/thyroidhormone superfamily of receptors selected from thyroid hormone receptorhomodimer, thyroid hormone receptor-retinoid X receptor heterodimer,retinoic acid receptor homodimer, or retinoic acid receptor-retinoid Xreceptor heterodimer,

[0086] a response element for said member of the steroid/thyroid hormonesuperfamily of receptors, wherein said response element is operativelylinked to a reporter, and

[0087] optionally, invention co-suppressor, and

[0088] wherein said second expression system comprises a complexcomprising:

[0089] a homodimeric or heterodimeric form of the same member of thesteroid/thyroid hormone superfamily of receptors as employed in saidfirst expression system, wherein said member is mutated such that itretains hormone dependent activation activity but has lost its abilityto repress basal level promoter activity of target genes,

[0090] the same response element-reporter combination as employed insaid first expression system, and

[0091] optionally, invention co-suppressor, and thereafter

[0092] selecting those compounds which provide:

[0093] increased reporter signal upon exposure of said compound to saidsecond expression system, relative to reporter signal in the absence ofsaid compound, and

[0094] substantially increased reporter signal upon exposure of saidcompound to said first expression system, relative to reporter signal inthe absence of said compound,

[0095] wherein said selected compounds are capable of relieving thesuppression of steroid/thyroid hormone receptor activity caused by aco-suppressor having a structure and function characteristic of thesilencing mediator for retinoic acid and thyroid receptors, andactivating said receptor.

[0096] In accordance with still another embodiment of the presentinvention, there are provided modified forms of the above-describedco-suppressor, including:

[0097] full length silencing mediator for retinoic acid and thyroidreceptors plus GAL4 DNA binding domain,

[0098] full length silencing mediator for retinoic acid and thyroidreceptors plus GAL4 activation domain,

[0099] full length silencing mediator for retinoic acid and thyroidreceptors plus glutathione S-transferase (GST) tag,

[0100] and the like.

[0101] The above-described modified forms of invention co-suppressor canbe used in a variety of ways, e.g., in the assays described herein.

[0102] An especially preferred modified co-suppressor of the inventioncomprises full length silencing mediator for retinoic acid and thyroidreceptors plus GAL4 activation domain.

[0103] In accordance with a still further embodiment of the presentinvention, there is provided a method to identify compounds whichdisrupt the ability of a co-suppressor as described hereinabove tocomplex with steroid/thyroid hormone receptors, without substantiallyactivating said receptor, said method comprising:

[0104] comparing the reporter signal produced by two differentexpression systems in the absence and presence of test compound,

[0105] wherein said first expression system comprises a complexcomprising:

[0106] a modified co-suppressor as described above,

[0107] a homodimeric or heterodimeric member of the steroid/thyroidhormone superfamily of receptors selected from thyroid hormone receptorhomodimer, thyroid hormone receptor-retinoid X receptor heterodimer,retinoic acid receptor homodimer or retinoic acid receptor-retinoid Xreceptor heterodimer, and

[0108] a response element for said member of the steroid/thyroid hormonesuperfamily of receptors, wherein said response element is operativelylinked to a reporter, and

[0109] wherein said second expression system comprises a complexcomprising:

[0110] said modified co-suppressor,

[0111] a homodimeric or heterodimeric form of the same member of thesteroid/thyroid hormone superfamily of receptors as employed in saidfirst expression system, wherein said member is mutated such that itretains hormone dependent activation activity but has lost its abilityto repress basal level promoter activity of target genes, and

[0112] the same response element-reporter combination as employed insaid first expression system, and thereafter

[0113] selecting those compounds which provide:

[0114] a lower reporter signal upon exposure of said compound to saidfirst expression system, relative to reporter signal in the absence ofsaid compound, and

[0115] substantially the same reporter signal upon exposure of saidcompound to said second expression system, relative to reporter signalin the absence of said compound,

[0116] wherein said selected compounds are capable of disrupting theability of a co-suppressor having a structure and functioncharacteristic of the silencing mediator for retinoic acid and thyroidreceptors to complex with steroid/thyroid hormone receptors, withoutsubstantially activating said receptor.

[0117] Mutant receptors contemplated for use in this embodiment of thepresent invention include RAR403 homodimers, RAR403-containingheterodimers, TR160 homodimers, TR160-containing heterodimers, and thelike.

[0118] Suitable host cells for use in this embodiment of the presentinvention include mammalian cells as well as yeast cells. Yeast cellsare presently preferred because they introduce no background since SMRT(i.e., silencing mediator (co-repressor) for retinoic acid receptor(RAR) and thyroid hormone receptor (TR)) is not endogenous to yeast.

[0119] In accordance with yet another embodiment of the presentinvention, there is provided a method to identify compounds whichactivate steroid/thyroid hormone receptor activity, but substantiallylack the ability to disrupt a complex comprising a steroid/thyroidhormone receptor and a co-suppressor as described hereinabove, saidmethod comprising:

[0120] comparing the reporter signal produced by two differentexpression systems in the absence and presence of test compound,

[0121] wherein said first expression system comprises a complexcomprising:

[0122] a modified co-suppressor as described above,

[0123] a homodimeric or heterodimeric member of the steroid/thyroidhormone superfamily of receptors selected from thyroid hormone receptorhomodimer, thyroid hormone receptor-retinoid X receptor heterodimer,retinoic acid receptor homodimer or retinoic acid receptor-retinoid Xreceptor heterodimer, and

[0124] a response element for said member of the steroid/thyroid hormonesuperfamily of receptors, wherein said response element is operativelylinked to a reporter, and

[0125] wherein said second receptor system comprises:

[0126] said modified co-suppressor,

[0127] a homodimeric or heterodimeric form of the same member of thesteroid/thyroid hormone superfamily of receptors as employed in saidfirst expression system, wherein said member is mutated such that itretains hormone dependent activation activity but has lost its abilityto repress basal level promoter activity of target genes, and

[0128] the same response element-reporter combination as employed insaid first expression system, and thereafter

[0129] selecting those compounds which provide:

[0130] a higher reporter signal upon exposure of said compound to saidsecond expression system, relative to reporter signal in the absence ofcompound, and

[0131] substantially the same reporter signal upon exposure of saidcompound to said first expression system, relative to reporter signal inthe absence of compound,

[0132] wherein said selected compounds are capable of activatingsteroid/thyroid hormone receptor activity, but substantially lack theability to disrupt the complex of a co-suppressor having a structure andfunction characteristic of the silencing mediator for retinoic acid andthyroid receptors and a steroid/thyroid hormone receptor.

[0133] Suitable host cells for use in this embodiment of the presentinvention include mammalian cells as well as yeast cells. Yeast cellsare presently preferred because they introduce no background since SMRTis not endogenous to yeast.

[0134] In accordance with a still further embodiment of the presentinvention, there is provided a method to identify compounds whichactivate a steroid/thyroid hormone receptor, and disrupt the ability ofa co-suppressor as described hereinabove to complex with said receptor,said method comprising:

[0135] comparing the reporter signal produced by two differentexpression systems in the absence and presence of test compound,

[0136] wherein said first expression system comprises a complexcomprising:

[0137] a modified co-suppressor as described above,

[0138] a homodimeric or heterodimeric member of the steroid/thyroidhormone superfamily of receptors selected from thyroid hormone receptorhomodimer, thyroid hormone receptor-retinoid X receptor heterodimer,retinoic acid receptor homodimer or retinoic acid receptor-retinoid Xreceptor heterodimer, and

[0139] a response element for said member of the steroid/thyroid hormonesuperfamily of receptors, wherein said response element is operativelylinked to a reporter, and

[0140] wherein said second expression system comprises a complexcomprising:

[0141] said modified co-suppressor,

[0142] the same homodimeric or heterodimeric member of thesteroid/thyroid hormone superfamily of receptors as employed in saidfirst expression system, wherein said member is mutated such that itretains hormone dependent activation activity but has lost its abilityto repress basal level promoter activity of target genes, and

[0143] the same response element-reporter combination as employed insaid first expression system, and thereafter

[0144] selecting those compounds which provide:

[0145] a reduction in reporter signal upon exposure of compound to saidfirst expression system, relative to reporter signal in the absence ofsaid compound, and

[0146] increased reporter signal upon exposure of compound to saidsecond expression system, relative to reporter signal in the absence ofsaid compound,

[0147] wherein said selected compounds are capable of activating asteroid/thyroid hormone receptor and disrupting a complex comprisingsteroid/thyroid hormone receptor and a co-suppressor having a structureand function characteristic of the silencing mediator for retinoic acidand thyroid receptors.

[0148] Suitable host cells for use in this embodiment of the presentinvention include mammalian cells as well as yeast cells. Yeast cellsare presently preferred because they introduce no background since SMRTis not endogenous to yeast.

[0149] In accordance with yet another aspect of the present invention,there is provided a method to identify compounds which activate asteroid/thyroid hormone receptor and/or disrupt the ability of aco-suppressor as described hereinabove to complex with said receptor,said method comprising:

[0150] comparing the reporter signals produced by a combinationexpression system in the absence and presence of test compound,

[0151] wherein said combination expression system comprises:

[0152] a first homodimeric or heterodimeric member of thesteroid/thyroid hormone superfamily of receptors selected from thyroidhormone receptor homodimer, thyroid hormone receptor-retinoid X receptorheterodimer, retinoic acid receptor homodimer, or retinoic acidreceptor-retinoid X receptor heterodimer,

[0153] a second homodimeric or heterodimeric form of the same member ofthe steroid/thyroid hormone superfamily of receptors as employed in saidfirst homodimer or heterodimer, wherein said member is mutated such thatit retains hormone dependent activation activity but has lost itsability to repress basal level promoter activity of target genes (i.e.,provides basal level expression),

[0154] wherein either said first homodimer (or heterodimer) or saidsecond homodimer (or heterodimer) is operatively linked to a GAL4 DNAbinding domain,

[0155] a response element for said member of the steroid/thyroid hormonesuperfamily of receptors, wherein said response element is operativelylinked to a first reporter,

[0156] a GAL4 response element, wherein said response element isoperatively linked to a second reporter, and

[0157] optionally a co-suppressor of steroid/thyroid hormone receptoractivity, said co-suppressor having a structure and functioncharacteristic of the silencing mediator for retinoic acid and thyroidreceptors, and thereafter

[0158] identifying as capable of relieving the suppression ofsteroid/thyroid hormone receptor activity caused by a co-suppressorhaving a structure and function characteristic of the silencing mediatorfor retinoic acid and thyroid receptors, but substantially lacking theability to activate steroid/thyroid hormone receptor activity thosecompounds which provide:

[0159] a higher reporter signal from the reporter responsive to thefirst member upon exposure of said compound to said first member,relative to reporter signal in the absence of said compound, and

[0160] substantially the same reporter signal from the reporterresponsive to the second member upon exposure of said compound to saidsecond member, relative to reporter signal in the absence of saidcompound, or

[0161] identifying as capable of activating steroid/thyroid hormonereceptor activity, but substantially lacking the ability to relieve thesuppression caused by a co-suppressor having a structure and functioncharacteristic of the silencing mediator for retinoic acid and thyroidreceptors those compounds which provide:

[0162] a higher reporter signal from the reporter responsive to thesecond member upon exposure of said compound to said second member,relative to reporter signal in the absence of compound, and

[0163] substantially the same reporter signal from the reporterresponsive to the first member upon exposure of said compound to saidfirst member, relative to reporter signal in the absence of saidcompound, or

[0164] identifying as capable of relieving the suppression ofsteroid/thyroid hormone receptor activity caused by a co-suppressorhaving a structure and function characteristic of the silencing mediatorfor retinoic acid and thyroid receptors, and activating said receptorthose compounds which provide:

[0165] a higher reporter signal from the reporter responsive to thesecond member upon exposure of said compound to said second member,relative to reporter signal in the absence of said compound, and

[0166] a greater increase in reporter signal from the reporterresponsive to the first member upon exposure of said compound to saidfirst member, relative to reporter signal in the absence of saidcompound.

[0167] Thus, the change in expression level of the two differentreporters introduced in a single transfection can be monitoredsimultaneously. Based on the results of this single transfection, onecan readily identify the mode of interaction of test compound with thereceptor/SMRT complex.

[0168] Exemplary GAL4 response elements are those containing thepalindromic 17-mer:

[0169] 5′-CGGAGGACTGTCCTCCG-3′ (SEQ ID NO:3),

[0170] such as, for example, 17MX, as described by Webster et al., inCell 52:169-178 (1988), as well as derivatives thereof. Additionalexamples of suitable response elements include those described byHollenberg and Evans in Cell 55:899-906 (1988); or Webster et al. inCell 54:199-207 (1988).

[0171] In accordance with still another embodiment of the presentinvention, there is provided a method to identify compounds whichactivate a steroid/thyroid hormone receptor and/or disrupt the abilityof a co-suppressor as described hereinabove to complex with saidreceptor, said method comprising:

[0172] comparing the reporter signals produced by a combinationexpression system in the absence and presence of test compound,

[0173] wherein said combination expression system comprises:

[0174] a modified co-suppressor as described above,

[0175] a first homodimeric or heterodimeric member of thesteroid/thyroid hormone superfamily of receptors selected from thyroidhormone receptor homodimer, thyroid hormone receptor-retinoid X receptorheterodimer, retinoic acid receptor homodimer, or retinoic acidreceptor-retinoid X receptor heterodimer,

[0176] a second homodimeric or heterodimeric form of the same member ofthe steroid/thyroid hormone superfamily of receptors as employed in saidfirst homodimer or heterodimer, wherein said member is mutated such thatit retains hormone dependent activation activity but has lost itsability to repress basal level promoter activity of target genes,

[0177] wherein either said first homodimer (or heterodimer) or saidsecond homodimer (or heterodimer) is operatively linked to a GAL4 DNAbinding domain,

[0178] a response element for said member of the steroid/thyroid hormonesuperfamily of receptors, wherein said response element is operativelylinked to a first reporter,

[0179] a GAL4 response element, wherein said response element isoperatively linked to a second reporter, and thereafter

[0180] identifying as capable of disrupting the ability of aco-suppressor having a structure and function characteristic of thesilencing mediator for retinoic acid and thyroid receptors to complexwith a steroid/thyroid hormone receptor, without substantiallyactivating steroid/thyroid hormone receptor, those compounds whichprovide:

[0181] a lower reporter signal from the reporter responsive to the firstmember upon exposure of said compound to said first member, relative toreporter signal in the absence of said compound, and

[0182] substantially the same reporter signal from the reporterresponsive to the second member upon exposure of said compound to saidsecond member, relative to reporter signal in the absence of saidcompound, or

[0183] identifying as capable of activating steroid/thyroid hormonereceptor activity, but substantially lacking the ability to disrupt acomplex comprising a steroid/thyroid hormone receptor and aco-suppressor having a structure and function characteristic of thesilencing mediator for retinoic acid and thyroid receptors, thosecompounds which provide:

[0184] a higher reporter signal from the reporter responsive to thesecond member upon exposure of said compound to said second member,relative to reporter signal in the absence of compound, and

[0185] substantially the same reporter signal from the reporterresponsive to the first member upon exposure of said compound to saidfirst member, relative to reporter signal in the absence of saidcompound, or

[0186] identifying as capable of disrupting a complex comprising asteroid/thyroid hormone receptor and a co-suppressor having a structureand function characteristic of the silencing mediator for retinoic acidand thyroid receptors, and activating said receptor those compoundswhich provide:

[0187] a reduction in reporter signal from the reporter responsive tothe first member upon exposure of said compound to said first member,relative to reporter signal in the absence of said compound, and

[0188] increased reporter signal from the reporter responsive to thesecond member upon exposure of said compound to said second member,relative to reporter signal in the absence of said compound.

[0189] In accordance with a still further aspect of the presentinvention, there is provided a method to identify compounds whichrelieve the suppression of steroid/thyroid hormone receptor activitycaused by a co-suppressor as described hereinabove, said methodcomprising determining the effect of adding test compound to anexpression system comprising:

[0190] a modified member of the steroid/thyroid hormone superfamily ofreceptors, wherein said modified member contains an activation domainwhich renders said receptor constitutively active,

[0191] a fusion protein comprising the receptor interaction domain ofSMRT operatively linked to the GAL4 DNA binding domain, and

[0192] a GAL4 response element operatively linked to a reporter.

[0193] Prior to addition of an effective ligand for the member of thesteroid/thyroid hormone superfamily of receptors employed herein, theassociation of the modified member and the fusion protein will beeffective to bind the GAL4 response element and activate transcriptionof the reporter. The presence of an effective ligand is indicated by areduction of reporter signal upon exposure to ligand, which disrupts theinteraction of the modified member and fusion protein.

[0194] Activation domains contemplated for use in the practice of thepresent invention are well known in the art and can readily beidentified by the artisan. Examples include the GAL4 activation domain,BP64, and the like.

[0195] To summarize, a novel nuclear receptor co-repressor whichmediates the transcriptional silencing of RAR and TR has beenidentified. This discovery is of great interest because transcriptionalsilencing has been shown to play an important role in development, celldifferentiation and the oncogenic activity of v-erbA (Baniahmad et al.,EMBO J. 11:1015-1023 (1992)); Gandrillon et al., Cell 49:687-697(1989)); Zenke et al., Cell 61:1035-1049 (1990); Barlow et al., EMBO J.13:4241-4250 (1994); Levine and Manley, Cell 59:405-408 (1989);Baniahmad et al., Proc. Natl. Acad. Sci. USA 89:10633-10637 (1992b) andSaitou et al., Nature 374:159-162 (1995)). In fact, v-erbA mutants thatharbor the Pro160->Arg change in the TR neither suppress basaltranscription nor are capable of oncogenic transformation (Damm andEvans, (1993) supra)

[0196] The function of SMRT as a silencing mediator (co-repressor) ofRAR and TR is analogous to mSin3 in the Mad-Max-Sin3 ternary complex(Schreiber-Agus et al., Cell 80:777-786 (1995); and Ayer et al., Cell80:767-776 (1995)). Because GAL-SMRT functions as a potent repressorwhen bound to DNA, it is reasonable to speculate that the function ofthe unliganded receptors is to bring with them SMRT to the template viaprotein-protein interaction. Thus, the repressor function is intrinsicto SMRT as opposed to the TR or RAR itself (Baniahmad et al., Proc.Natl. Acad. Sci. USA 90:8832-8836 (1993); and Fondell et al., Genes Dev7:1400-1410 (1993)). It is demonstrated herein that the ligand triggersa dissociation of SMRT from the receptor, which would lead to an initialstep in the activation process. This would be followed (or becoincident) with an induced conformational change in thecarboxy-terminal transactivation domain (τc, also called AF-2), allowingassociation with co-activators on the transcription machinery (Douarinet al., EMBO J. 14:2020-2033 (1995); Halachmi et al., Science264:1455-1458 (1994); Lee et al., Nature 374:91-94 (1995); and Cavailleset al., Proc. Natl. Acad. Sci. USA 91:10009-10013 (1994)). Thus, as haspreviously been suggested (Damm and Evans (1993) supra), the liganddependent activation of TR would represent two separable processesincluding relief of repression and net activation. The isolation of SMRTnow provides a basis for dissecting the molecular basis oftrans-repression.

[0197] The invention will now be described in greater detail byreference to the following non-limiting examples.

EXAMPLE 1 Isolation of SMRT

[0198] Using a GAL4 DBD-RXR fusion protein (see, for example, U.S. Ser.No. 08/177,740, incorporated by reference herein in its entirety) as abait in a yeast two-hybrid screening system (Durfee et al. Genes Dev7:555-569 (1993)), several cDNA clones encoding receptor interactingproteins were isolated. One of these proteins, SMRT, interacts stronglywith unliganded RAR and TR but only weakly with RXR or other receptorsin yeast. This protein was selected for further characterization.

EXAMPLE 2 Far-Western Blotting Procedure

[0199] Total bacteria extracts expressing GST fusions of hRARα (aa156-462) or hRXRα LBD (aa 228-462) and control extracts expressing GSTalone or GST-PML fusion protein were subjected to SDS/PAGE andelectroblotted onto nitrocellulose in transfer buffer (25 mM Tris, pH8.3/192 mM glycine/0.01% SDS). After denaturation/renaturation from 6 Mto 0.187 M guanidine hydrochloride in HB buffer (25 mM Hepes, pH 7.7/25mM NaCl/5 mM MgCl₂/1 mM DTT) filters were saturated at 4° C. in blockingbuffer (5% milk, then 1% milk in HB buffer plus 0.05% NONIDET® P-40(nonylphenol polyoxyethylene ether)). In vitro translated ³⁵S-labeledproteins were diluted into H buffer (20 mM Hepes, pH 7.7/75 mM KCl/0.1mM EDTA/2.5 mM MgCl₂/0.05% NONIDET® P-40 (nonylphenol polyoxyethyleneether)/1% milk/1 mM DTT) and the filters were hybridized overnight at 4°C. with (1 μM) or without ligand. After three washes with H buffer,filters were dried and exposed for autoradiography or quantitated byphosphoimager.

[0200] GST-SMRT is a GST fusion of the C-SMRT encoded by the yeast twohybrid clone. GST-SMRT has been purified, but contains severaldegradation products.

[0201] For yeast two-hybrid screening, a construct expressing the GAL4DBD-hRXRα LBD (aa 198-462) fusion protein was used to screen a humanlymphocyte cDNA library as described (Durfee et al., (1993) supra). Fulllength SMRT cDNA was isolated from a human HeLa cDNA library (Clontech)using the two-hybrid insert as a probe.

[0202] Using the above-described far-western blotting procedure,³⁵S-labeled SMRT preferentially complexes with bacterial extractsexpressing the RAR, marginally associates with RXR and shows noassociation with control extracts. In contrast, ³⁵S-PPAR selectivelyassociates with its heterodimeric partner, RXR, but not with RAR. In asimilar assay, ³⁵S-labeled RAR or TR interacts strongly with SMRT andtheir heterodimeric partner, RXR, but not with degraded GST products,while ³⁵S-RXR interacts only weakly with SMRT. Binding of ligand to RARor TR reduces their interactions with SMRT but not with RXR, whilebinding of ligand to RXR has only slight effect. FIG. 1 shows thequantitation of a dose-dependent dissociation of SMRT from RAR or TR byall-trans retinoic acid (atRA) or thyroid hormone (triiodothyronine orT3), demonstrating that the amount of ligand required for 50%dissociation in both cases are close to the kds for both ligands (Munozet al. EMBO J. 7:155-159 (1988); Sap et al., Nature 340:242-244 (1989);and Yang et al., Proc. Natl. Acad. Sci. USA 88:3559-3563 (1991)).

[0203] Full length SMRT encodes a polypeptide of 1495 amino acids richin proline and serine residues (see FIG. 2 and SEQ ID NO:1). Genbankdatabase comparison reveals similarity of the C-terminal domain of SMRTto a partial cDNA encoding another receptor interacting protein, RIP13(Seol et al., (1995) supra), whose role in receptor signaling isunknown. Within this region, there can be identified several potentialheptad repeats which might mediate protein-protein interaction with the“a-helical sandwich” structure (Bourguet et al., Nature 375:377-382(1995)) of the ligand binding domain (LBD) of receptors.

EXAMPLE 3 Characterization of SMRT

[0204] Unlike other nuclear receptors, unliganded RAR and TR possess astrong silencing domain which represses basal level promoter activity oftheir target genes (Damm et al., Nature 339:593-597 (1989); Brent etal., New Biol. 1:329-336 (1989); Baniahmad et al., Cell 61:505-514(1990); and Baniahmad et al., EMBO J. 11:1015-1023 (1992)). Thepreferential interaction of SMRT with RAR and TR in the absence ofhormone suggests that SMRT may play a role in mediating thetranscriptional silencing effect of the receptor.

[0205] To further investigate the involvement of SMRT in silencing, theinteraction of SMRT with mutant receptors which display distinctsilencing and/or transactivation activities was tested as follows.³⁵S-methionine labeled receptors were used as probes to hybridizeimmobilized GST-SMRT in the presence (10 μM) or absence of all-transretinoic acid (atRA). The total bacteria extract expressing GST-RXR wasincluded as a control.

[0206] When quantitated by phosphoimager, RAR403 shows a 4-fold betterinteraction with SMRT than wild type RAR. Both full length RAR or adeletion mutant expressing only the ligand binding domain (LBD, referredto as ΔΔR) associate with SMRT; this association is blocked by ligand.

[0207] These results confirm that the LBD alone is sufficient in theinteraction. The carboxy-terminal deletion mutant RAR403 is a potentdominant negative suppressor of basal level promoter activity of RARtarget genes (Damm et al., Proc. Natl. Acad. Sci. USA 90:2989-2993(1993); Tsai and Collins, Proc. Natl. Acad. Sci. USA 90:7153-7157(1993); and Tsai et al., Genes Dev 6:2258-2269 (1992)). As might bepredicted from the above studies, RAR403 and its amino terminal deletionderivative, ΔΔR403, interact strongly with SMRT in either the presenceor absence of ligand, consistent with SMRT mediating the repressoractivity of this mutant.

EXAMPLE 4 Interaction of SMRT with TR Mutants

[0208] The interaction of SMRT with two different classes of TR mutantswas analyzed next. The first mutant employed is the naturally occurringoncogene, v-erbA, which has strong silencing ability but notransactivation activity (Sap et al., (1989) supra Sap et al., Nature324:635-640 (1986); Weinberger et al., Nature 318:670-672 (1985); andWeinberger et al., Nature 324:641-646 (1986)). The second mutantemployed is a single amino acid change (Pro 160->Arg) of the rTRa(TR160) which has previously been shown to lose its capacity in basallevel suppression but retains hormone dependent transactivation(Thompson et al., Science 237:1610-1614 (1987); and Damm and Evans,Proc. Natl. Acad. Sd. USA 90:10668-10672 (1993)). If SMRT is involved insilencing, it would be expected that SMRT should interact with thev-erbA, but show little or no association with the silencing-defectiveTR160 mutant.

[0209] Interaction of the oncogenic v-erbA and rTRα R160 mutant (TR160)with GST-SMRT was determined in a far-western assay as described above(see Example 2). When quantitated by phosphoimager, the v-erbA shows an18-fold better interaction with SMRT than hTRβ, and the TR160 mutantshows a 10-fold lower signal than the rTRα.

[0210] As one might expect, v-erbA interacts strongly with SMRT both inpresence or absence of ligand. In contrast, full length TR160 mutant orLBD of TR160 ΔΔTR160) does not interact significantly with SMRT whencompared to the wild type receptor.

[0211] These data demonstrate that SMRT plays an important role inmediating transcriptional silencing effects of both RAR and TR. Thesedata also suggest that the release of SMRT from receptors could be aprerequisite step in ligand-dependent transactivation by nuclearreceptors.

EXAMPLE 5 Formation of Ternary Complexes Containing SMRT

[0212] RAR and TR form heterodimers with RXR, resulting in a complexwith high DNA binding ability (Bugge et al., EMBO J. 11:1409-1418(1992); Yu et al., Cell 67:1251-1266 (1991); and Kliewer et al., Nature355:446-449 (1992)). Since SMRT interacts with RAR and TR, tests wereconducted to determine whether SMRT can also interact with thereceptor-DNA complex. Thus, the interaction of SMRT with RXR-RARheterodimer on a DR5 element (i.e., an AGGTCA direct repeat spaced byfive nucleotides) was determined in a gel retardation assay, which iscarried out as follows. In vitro translated receptor or unprogrammedreticulocyte lysate (URL) was incubated with 1 μg of poly dIdC on icefor 15 minutes in a total volume of 20 μl containing 75 mM KCl, 7.5%glycerol, 20 mM Hepes (pH 7.5), 2 mM DTT and 0.1% NONIDET® P-40(nonylphenol polyoxyethylene ether), with or without ligand (in therange of about 10-100 nM employed). A ³²P labeled, double strandedoligonucleotide probe was added into the binding reaction (10,000 cpmper reaction), and the reaction was further incubated for 20 minutes atroom temperature. The protein-DNA complex was separated on a 5% nativepolyacrylamide gel at 150 volts.

[0213] SMRT is seen to form a ternary complex with the RXR-RARheterodimer on a DNA response element in the gel retardation assay.Addition of ligand releases SMRT from this complex in a dose-dependentmanner.

[0214] Similarly, SMRT is seen to form a ternary complex with the RXR-TRheterodimer on a TR response element; addition of T3 disrupts theformation of this complex.

[0215] These data demonstrate that SMRT can be recruited to DNA responseelements via protein-protein interaction with RAR or TR in the absenceof hormone. Binding of hormone disrupts receptor-SMRT interaction andreleases SMRT from the receptor-DNA complex.

EXAMPLE 6 Transient Transfection Assay

[0216] CV-1 cells were plated in 24 well plates at a density of 50,000cells per well. Expression plasmids were transfected into cells bylipofection using DOTAP. In each transfection, 5 ng of GAL-RAR and 15 ngof v-erbA or SMRT were used together with 150 ng of reporter constructcontaining 4 copies of GAL4 binding sites in front of a minimalthymidine kinase promoter and a CMX-β-gal construct as an internalcontrol. The relative luciferase activity was calculated by normalizingto the β-gal activity.

EXAMPLE 7 Reversal of Transcriptional Silencing

[0217] Recently, it has been shown that over expression of RAR or TRcould reverse the transcriptional silencing effect of the GAL4 DBDfusion of TR (GAL-TR) or RAR (GAL-RAR) (Baniahmad et al., Mol Cell Biol15:76-86 (1995); and Casanova et al., Mol Cell Biol. 14:5756-5765(1994)), co-repressor. A similar effect is observed herein when overexpression of v-erbA or RAR403 mutants are shown to reverse thesilencing effect of GAL-RAR and GAL-TR on the basal activity of aluciferase reporter (see FIGS. 3A and 3B).

[0218] In principle, over expression of SMRT should restore repressoractivity when co-expressed with v-erbA or RAR403 competitors. Indeed,results presented in FIG. 3C show that both the full length and theC-terminal domain of SMRT (C-SMRT) can titrate out v-erbA or RAR403competitor activity and re-endow GAL-RAR and GAL-TR with silencingactivity. In contrast, neither v-erbA nor SMRT show any effect on thetransactivation activity of GAL-VP16 fusion. Thus, SMRT is able to blockthe titration effect of v-erbA and RAR403 and functionally replaces theputative co-repressor in this system.

EXAMPLE 8 Direct Recruitment of SMRT to a Heterologous Promoter

[0219] If SMRT is the mediator of transcription silencing of TR and RARby interaction with template-bound unliganded receptors, then directrecruitment of SMRT to a heterologous promoter should result inrepression of basal level activity. This was tested by fusing fulllength SMRT to the GAL4 DBD (GAL-SMRT). The effect of the resultingfusion protein on the activity of the thymidine kinase promotercontaining four GAL4 binding sites was analyzed. FIG. 3D shows thatGAL-SMRT, like GAL-TR, can silence basal promoter activity in adose-dependent manner. In contrast, GAL-RXR shows no suppression.

[0220] These data suggest that SMRT, when recruited to a promoter bydirect DNA binding or via association with an unliganded receptor,functions as a potent transcriptional repressor.

[0221] While the invention has been described in detail with referenceto certain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed. SEQUENCE ID NO:1 1 MEAWDAH

DKEAFAAEAQKL

GD

CWTSGL

F

V

REVIKAS

HA

D

51 SAFSYA

GH

L

LGLHDTAR

VL

R

TISN

LISSAKH

SVLERQI 101 GAISQGMSVQLHV

YSEHAKA

VQ

VTMGL

L

MD

KKLA

FSGVKQEQL 151 SPRGQAGPPESLGVPTAQEASVLRGTALGSVPGGSITKGIPSTRVPSDSA 201ITYRGSITHGTPADVLYKGTITRIIGEDSPSRLDRGREDSLPKGHVIYEG 251KKGHVLSYEGGMSVTQCSKEDGRSSSGPPHETAAPKRTYDMMEGRVGRAI 301SSASIEGLMGRAIPPERHSPHHLKEQHHIRGSITQGIPRSYVEAQEDYLR 351REAKLLKREGTPPPPPPSRDLTEAYKTQALGPLKLKPAHEGLVATVKEAG 401RSIHEIPREELRHTPELPLAPRPLKEGSITQGTPLKYDTGASTTGSKKHD 451VRSLIGSPGRTFPPVHPLDVMADARALERACYEESLKSRPGTASSSGGSI 501ARGAPVIVPELGKPRQSPLTYEDHGAPFAGHLPRGSPVTMREPTPRLQEG 551SLSSSKASQDRKLTSTPREIAKSPHSTVPEHHPHPISPYEHLLRGVSGVD 601LYRSHIPLAFDPTSIPRGIPLDAAAAYYLPRHLAPNPTYPHLYPPYLIRG 651YPDTAALENRQTIINDYITSQQMHHNTATAMAQRADMLRGLSPRESSLAL 701NYAAGPRGIIDLSQVPHLPVLVPPTPGTPATAMDRLAYLPTAPQPFSSRH 751SSSPLSPGGPTHLTKPTTTSSSERERDRDRERDRDREREKSILTSTTTVE 801HAPIWRPGTEQSSGSSGSSGGGGGSSSRPASHSHAHQHSPISPRTQDALQ 851QRPSVLHNTGMKGIITAVEPSKPTVLRSTSTSSPVRPAATFPPATHCPLG 901GTLDGVYPTLMEPVLLPKEAPRVARPERPRADTGHAFLAKPPARSGLEPA                               |{right arrow over (   )}C-SMRT 951SSPSKGSEPRPLVPPVSGHATIARTPAKNLAPHHASPDPPAPPASASDPH 1001REKTQSKPFSIQELELRSLGYHGSSYSPEGVEPVSPVSSPSLTHDKGLPK 1051HLEELDKSHLEGELRPKQPGPVKLGGEAAHLPHLRPLPES

PSSSPLL

T 1101 APGVKGH

RVVTLA

HISEVIT

DYTRHHP

LSAPLPAPLYSFPGASCP 1151VLDLRRPPSDLYLPPPDHGAPARGSPHSEGGKRSPEPNKTSVLGGGEDGI 1201EPVSPPEGMTEPGHSRSAVYPLLYRDGEQTEPSRMGSKSPGNTSQPPAFF 1251 SK

TESNSA

KSKKQE

NKKLNT

NRNEPEYNISQPGTEIFNMPAITGT 1301 GL

TYRSQA

QEHASTNMGLEAIIRKALM┌GKYDQW.EESPPLSANAFNPL 1350NASASLPAAMPITAADGRSDHTLTSP┐.GGGGKAKVSGRPSSRKAKSPAPG 1399LA..SGDRPPSVSSVHSEGDCNRRTPLTNRVWEDRPSSAGSTPFPYNPL

1447 MRLQAG

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PAGSGP

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CSQYET

1492 SDSE*  1495 SEQUENCE ID NO:2GKYDQWEESPPLSANAFNPLNASASLPAAMPITAADGRSDHTLTSP

[0222] SEQUENCE ID NO:2

[0223] GKYDQWEESPPLSANAFNPLNASASLPAAMPITAADGRSDHTLTSP

1 3 1 1495 PRT Homo sapiens 1 Met Glu Ala Trp Asp Ala His Pro Asp LysGlu Ala Phe Ala Ala Glu 1 5 10 15 Ala Gln Lys Leu Pro Gly Asp Pro ProCys Trp Thr Ser Gly Leu Pro 20 25 30 Phe Pro Val Pro Pro Arg Glu Val IleLys Ala Ser Pro His Ala Pro 35 40 45 Asp Pro Ser Ala Phe Ser Tyr Ala ProPro Gly His Pro Leu Pro Leu 50 55 60 Gly Leu His Asp Thr Ala Arg Pro ValLeu Pro Arg Pro Pro Thr Ile 65 70 75 80 Ser Asn Pro Pro Pro Leu Ile SerSer Ala Lys His Pro Ser Val Leu 85 90 95 Glu Arg Gln Ile Gly Ala Ile SerGln Gly Met Ser Val Gln Leu His 100 105 110 Val Pro Tyr Ser Glu His AlaLys Ala Pro Val Gly Pro Val Thr Met 115 120 125 Gly Leu Pro Leu Pro MetAsp Pro Lys Lys Leu Ala Pro Phe Ser Gly 130 135 140 Val Lys Gln Glu GlnLeu Ser Pro Arg Gly Gln Ala Gly Pro Pro Glu 145 150 155 160 Ser Leu GlyVal Pro Thr Ala Gln Glu Ala Ser Val Leu Arg Gly Thr 165 170 175 Ala LeuGly Ser Val Pro Gly Gly Ser Ile Thr Lys Gly Ile Pro Ser 180 185 190 ThrArg Val Pro Ser Asp Ser Ala Ile Thr Tyr Arg Gly Ser Ile Thr 195 200 205His Gly Thr Pro Ala Asp Val Leu Tyr Lys Gly Thr Ile Thr Arg Ile 210 215220 Ile Gly Glu Asp Ser Pro Ser Arg Leu Asp Arg Gly Arg Glu Asp Ser 225230 235 240 Leu Pro Lys Gly His Val Ile Tyr Glu Gly Lys Lys Gly His ValLeu 245 250 255 Ser Tyr Glu Gly Gly Met Ser Val Thr Gln Cys Ser Lys GluAsp Gly 260 265 270 Arg Ser Ser Ser Gly Pro Pro His Glu Thr Ala Ala ProLys Arg Thr 275 280 285 Tyr Asp Met Met Glu Gly Arg Val Gly Arg Ala IleSer Ser Ala Ser 290 295 300 Ile Glu Gly Leu Met Gly Arg Ala Ile Pro ProGlu Arg His Ser Pro 305 310 315 320 His His Leu Lys Glu Gln His His IleArg Gly Ser Ile Thr Gln Gly 325 330 335 Ile Pro Arg Ser Tyr Val Glu AlaGln Glu Asp Tyr Leu Arg Arg Glu 340 345 350 Ala Lys Leu Leu Lys Arg GluGly Thr Pro Pro Pro Pro Pro Pro Ser 355 360 365 Arg Asp Leu Thr Glu AlaTyr Lys Thr Gln Ala Leu Gly Pro Leu Lys 370 375 380 Leu Lys Pro Ala HisGlu Gly Leu Val Ala Thr Val Lys Glu Ala Gly 385 390 395 400 Arg Ser IleHis Glu Ile Pro Arg Glu Glu Leu Arg His Thr Pro Glu 405 410 415 Leu ProLeu Ala Pro Arg Pro Leu Lys Glu Gly Ser Ile Thr Gln Gly 420 425 430 ThrPro Leu Lys Tyr Asp Thr Gly Ala Ser Thr Thr Gly Ser Lys Lys 435 440 445His Asp Val Arg Ser Leu Ile Gly Ser Pro Gly Arg Thr Phe Pro Pro 450 455460 Val His Pro Leu Asp Val Met Ala Asp Ala Arg Ala Leu Glu Arg Ala 465470 475 480 Cys Tyr Glu Glu Ser Leu Lys Ser Arg Pro Gly Thr Ala Ser SerSer 485 490 495 Gly Gly Ser Ile Ala Arg Gly Ala Pro Val Ile Val Pro GluLeu Gly 500 505 510 Lys Pro Arg Gln Ser Pro Leu Thr Tyr Glu Asp His GlyAla Pro Phe 515 520 525 Ala Gly His Leu Pro Arg Gly Ser Pro Val Thr MetArg Glu Pro Thr 530 535 540 Pro Arg Leu Gln Glu Gly Ser Leu Ser Ser SerLys Ala Ser Gln Asp 545 550 555 560 Arg Lys Leu Thr Ser Thr Pro Arg GluIle Ala Lys Ser Pro His Ser 565 570 575 Thr Val Pro Glu His His Pro HisPro Ile Ser Pro Tyr Glu His Leu 580 585 590 Leu Arg Gly Val Ser Gly ValAsp Leu Tyr Arg Ser His Ile Pro Leu 595 600 605 Ala Phe Asp Pro Thr SerIle Pro Arg Gly Ile Pro Leu Asp Ala Ala 610 615 620 Ala Ala Tyr Tyr LeuPro Arg His Leu Ala Pro Asn Pro Thr Tyr Pro 625 630 635 640 His Leu TyrPro Pro Tyr Leu Ile Arg Gly Tyr Pro Asp Thr Ala Ala 645 650 655 Leu GluAsn Arg Gln Thr Ile Ile Asn Asp Tyr Ile Thr Ser Gln Gln 660 665 670 MetHis His Asn Thr Ala Thr Ala Met Ala Gln Arg Ala Asp Met Leu 675 680 685Arg Gly Leu Ser Pro Arg Glu Ser Ser Leu Ala Leu Asn Tyr Ala Ala 690 695700 Gly Pro Arg Gly Ile Ile Asp Leu Ser Gln Val Pro His Leu Pro Val 705710 715 720 Leu Val Pro Pro Thr Pro Gly Thr Pro Ala Thr Ala Met Asp ArgLeu 725 730 735 Ala Tyr Leu Pro Thr Ala Pro Gln Pro Phe Ser Ser Arg HisSer Ser 740 745 750 Ser Pro Leu Ser Pro Gly Gly Pro Thr His Leu Thr LysPro Thr Thr 755 760 765 Thr Ser Ser Ser Glu Arg Glu Arg Asp Arg Asp ArgGlu Arg Asp Arg 770 775 780 Asp Arg Glu Arg Glu Lys Ser Ile Leu Thr SerThr Thr Thr Val Glu 785 790 795 800 His Ala Pro Ile Trp Arg Pro Gly ThrGlu Gln Ser Ser Gly Ser Ser 805 810 815 Gly Ser Ser Gly Gly Gly Gly GlySer Ser Ser Arg Pro Ala Ser His 820 825 830 Ser His Ala His Gln His SerPro Ile Ser Pro Arg Thr Gln Asp Ala 835 840 845 Leu Gln Gln Arg Pro SerVal Leu His Asn Thr Gly Met Lys Gly Ile 850 855 860 Ile Thr Ala Val GluPro Ser Lys Pro Thr Val Leu Arg Ser Thr Ser 865 870 875 880 Thr Ser SerPro Val Arg Pro Ala Ala Thr Phe Pro Pro Ala Thr His 885 890 895 Cys ProLeu Gly Gly Thr Leu Asp Gly Val Tyr Pro Thr Leu Met Glu 900 905 910 ProVal Leu Leu Pro Lys Glu Ala Pro Arg Val Ala Arg Pro Glu Arg 915 920 925Pro Arg Ala Asp Thr Gly His Ala Phe Leu Ala Lys Pro Pro Ala Arg 930 935940 Ser Gly Leu Glu Pro Ala Ser Ser Pro Ser Lys Gly Ser Glu Pro Arg 945950 955 960 Pro Leu Val Pro Pro Val Ser Gly His Ala Thr Ile Ala Arg ThrPro 965 970 975 Ala Lys Asn Leu Ala Pro His His Ala Ser Pro Asp Pro ProAla Pro 980 985 990 Pro Ala Ser Ala Ser Asp Pro His Arg Glu Lys Thr GlnSer Lys Pro 995 1000 1005 Phe Ser Ile Gln Glu Leu Glu Leu Arg Ser LeuGly Tyr His Gly 1010 1015 1020 Ser Ser Tyr Ser Pro Glu Gly Val Glu ProVal Ser Pro Val Ser 1025 1030 1035 Ser Pro Ser Leu Thr His Asp Lys GlyLeu Pro Lys His Leu Glu 1040 1045 1050 Glu Leu Asp Lys Ser His Leu GluGly Glu Leu Arg Pro Lys Gln 1055 1060 1065 Pro Gly Pro Val Lys Leu GlyGly Glu Ala Ala His Leu Pro His 1070 1075 1080 Leu Arg Pro Leu Pro GluSer Gln Pro Ser Ser Ser Pro Leu Leu 1085 1090 1095 Gln Thr Ala Pro GlyVal Lys Gly His Gln Arg Val Val Thr Leu 1100 1105 1110 Ala Gln His IleSer Glu Val Ile Thr Gln Asp Tyr Thr Arg His 1115 1120 1125 His Pro GlnGln Leu Ser Ala Pro Leu Pro Ala Pro Leu Tyr Ser 1130 1135 1140 Phe ProGly Ala Ser Cys Pro Val Leu Asp Leu Arg Arg Pro Pro 1145 1150 1155 SerAsp Leu Tyr Leu Pro Pro Pro Asp His Gly Ala Pro Ala Arg 1160 1165 1170Gly Ser Pro His Ser Glu Gly Gly Lys Arg Ser Pro Glu Pro Asn 1175 11801185 Lys Thr Ser Val Leu Gly Gly Gly Glu Asp Gly Ile Glu Pro Val 11901195 1200 Ser Pro Pro Glu Gly Met Thr Glu Pro Gly His Ser Arg Ser Ala1205 1210 1215 Val Tyr Pro Leu Leu Tyr Arg Asp Gly Glu Gln Thr Glu ProSer 1220 1225 1230 Arg Met Gly Ser Lys Ser Pro Gly Asn Thr Ser Gln ProPro Ala 1235 1240 1245 Phe Phe Ser Lys Leu Thr Glu Ser Asn Ser Ala MetVal Lys Ser 1250 1255 1260 Lys Lys Gln Glu Ile Asn Lys Lys Leu Asn ThrHis Asn Arg Asn 1265 1270 1275 Glu Pro Glu Tyr Asn Ile Ser Gln Pro GlyThr Glu Ile Phe Asn 1280 1285 1290 Met Pro Ala Ile Thr Gly Thr Gly LeuMet Thr Tyr Arg Ser Gln 1295 1300 1305 Ala Val Gln Glu His Ala Ser ThrAsn Met Gly Leu Glu Ala Ile 1310 1315 1320 Ile Arg Lys Ala Leu Met GlyLys Tyr Asp Gln Trp Glu Glu Ser 1325 1330 1335 Pro Pro Leu Ser Ala AsnAla Phe Asn Pro Leu Asn Ala Ser Ala 1340 1345 1350 Ser Leu Pro Ala AlaMet Pro Ile Thr Ala Ala Asp Gly Arg Ser 1355 1360 1365 Asp His Thr LeuThr Ser Pro Gly Gly Gly Gly Lys Ala Lys Val 1370 1375 1380 Ser Gly ArgPro Ser Ser Arg Lys Ala Lys Ser Pro Ala Pro Gly 1385 1390 1395 Leu AlaSer Gly Asp Arg Pro Pro Ser Val Ser Ser Val His Ser 1400 1405 1410 GluGly Asp Cys Asn Arg Arg Thr Pro Leu Thr Asn Arg Val Trp 1415 1420 1425Glu Asp Arg Pro Ser Ser Ala Gly Ser Thr Pro Phe Pro Tyr Asn 1430 14351440 Pro Leu Ile Met Arg Leu Gln Ala Gly Val Met Ala Ser Pro Pro 14451450 1455 Pro Pro Gly Leu Pro Ala Gly Ser Gly Pro Leu Ala Gly Pro His1460 1465 1470 His Ala Trp Asp Glu Glu Pro Lys Pro Leu Leu Cys Ser GlnTyr 1475 1480 1485 Glu Thr Leu Ser Asp Ser Glu 1490 1495 2 46 PRTARTIFICIAL RESIDUES 1330-1376 OF SEQUENCE 1 2 Gly Lys Tyr Asp Gln TrpGlu Glu Ser Pro Pro Leu Ser Ala Asn Ala 1 5 10 15 Phe Asn Pro Leu AsnAla Ser Ala Ser Leu Pro Ala Ala Met Pro Ile 20 25 30 Thr Ala Ala Asp GlyArg Ser Asp His Thr Leu Thr Ser Pro 35 40 45 3 17 DNA ARTIFICIAL GAL4RESPONSE ELEMENT palindromic 3 cggaggactg tcctccg 17

That which is claimed is:
 1. A method to identify compounds whichrelieve the suppression of steroid/thyroid hormone receptor activitycaused by a co-suppressor of steroid/thyroid hormone receptor activity,said co-suppressor having a structure and function characteristic of thesilencing mediator for retinoic acid and thyroid hormone receptors,without substantially activating said receptor, said method comprising:comparing the reporter signal produced by two different expressionsystems in the absence and presence of test compound, wherein said firstexpression system comprises a complex comprising: a homodimeric orheterodimeric member of the steroid/thyroid hormone superfamily ofreceptors selected from thyroid hormone receptor homodimer, thyroidhormone receptor-retinoid X receptor heterodimer, retinoic acid receptorhomodimer, or retinoic acid receptor-retinoid X receptor heterodimer, aresponse element for said member of the steroid/thyroid hormonesuperfamily of receptors, wherein said response element is operativelylinked to a reporter, and optionally, said co-suppressor, and whereinsaid second expression system comprises a complex comprising: ahomodimeric or heterodimeric form of the same member of thesteroid/thyroid hormone superfamily of receptors as employed in saidfirst expression system, wherein said member is mutated such that itretains hormone dependent activation activity but has lost its abilityto repress basal level promoter activity of target genes, the sameresponse element-reporter combination as employed in said firstexpression system, and optionally, said co-suppressor, and thereafterselecting those compounds which provide: a higher reporter signal uponexposure of said compound to said first expression system, relative toreporter signal in the absence of said compound, and substantially thesame reporter signal upon exposure of said compound to said secondexpression system, relative to reporter signal in the absence of saidcompound, wherein said selected compounds are capable of relieving thesuppression of steroid/thyroid hormone receptor activity caused by aco-suppressor having a structure and function characteristic of thesilencing mediator for retinoic acid and thyroid hormone receptors, butsubstantially lacking the ability to activate steroid/thyroid hormonereceptor activity.
 2. A method according to claim 1 wherein said mutantreceptor is selected from RAR403 homodimers, RAR403-containingheterodimers, TR160 homodimers or TR160-containing heterodimers.
 3. Amethod to identify compounds which activate steroid/thyroid hormonereceptor activity, but substantially lack the ability to relieve thesuppression caused by a co-suppressor of steroid/thyroid hormonereceptor activity, said co-suppressor having a structure and functioncharacteristic of the silencing mediator for retinoic acid and thyroidhormone receptors, said method comprising: comparing the reporter signalproduced by two different expression systems in the absence and presenceof test compound, wherein said first expression system comprises acomplex comprising: a homodimeric or heterodimeric member of thesteroid/thyroid hormone superfamily of receptors selected from the groupconsisting of thyroid hormone receptor homodimer, thyroid hormonereceptor-retinoid X receptor heterodimer, retinoic acid receptorhomodimer, and retinoic acid receptor-retinoid X receptor heterodimer, aresponse element for said member of the steroid/thyroid hormonesuperfamily of receptors, wherein said response element is operativelylinked to a reporter, and optionally, said co-suppressor, and whereinsaid second expression system comprises a complex comprising: ahomodimeric or heterodimeric form of the same member of thesteroid/thyroid hormone superfamily of receptors as employed in saidfirst expression system, wherein said member is mutated such that itretains hormone dependent activation activity but has lost its abilityto repress basal level promoter activity of target genes, the sameresponse element-reporter combination as employed in said firstexpression system, and optionally, said co-suppressor, and thereafterselecting those compounds which provide: a higher reporter signal uponexposure of said compound to said second expression system, relative toreporter signal in the absence of compound, and substantially the samereporter signal upon exposure of said compound to said first expressionsystem, relative to reporter signal in the absence of said compound,wherein said selected compounds are capable of activatingsteroid/thyroid hormone receptor activity, but substantially lacking theability to relieve the suppression caused by a co-suppressor having astructure and function characteristic of the silencing mediator forretinoic acid and thyroid hormone receptors.
 4. A method to identifycompounds which relieve the suppression of steroid/thyroid hormonereceptor activity caused by a co-suppressor having a structure andfunction characteristic of the silencing mediator for retinoic acid andthyroid hormone receptors, and activate said receptor, said methodcomprising: comparing the reporter signal produced by two differentexpression systems in the absence and presence of test compound, whereinsaid first expression system comprises a complex comprising: ahomodimeric or heterodimeric member of the steroid/thyroid hormonesuperfamily of receptors selected from thyroid hormone receptorhomodimer, thyroid hormone receptor-retinoid X receptor heterodimer,retinoic acid receptor homodimer, or retinoic acid receptor-retinoid Xreceptor heterodimer, a response element for said member of thesteroid/thyroid hormone superfamily of receptors, wherein said responseelement is operatively linked to a reporter, and optionally, saidco-suppressor, and wherein said second expression system comprises acomplex comprising: a homodimeric or heterodimeric form of the samemember of the steroid/thyroid hormone superfamily of receptors asemployed in said first expression system, wherein said member is mutatedsuch that it retains hormone dependent activation activity but has lostits ability to repress basal level promoter activity of target genes,the same response element-reporter combination as employed in said firstexpression system, and optionally, said co-suppressor, and thereafterselecting those compounds which provide: increased reporter signal uponexposure of said compound to said second expression system, relative toreporter signal in the absence of said compound, and substantiallyincreased reporter signal upon exposure of said compound to said firstexpression system, relative to reporter signal in the absence of saidcompound, wherein said selected compounds are capable of relieving thesuppression of steroid/thyroid hormone receptor activity caused by aco-suppressor of steroid/thyroid hormone receptor activity, saidco-suppressor having a structure and function characteristic of thesilencing mediator for retinoic acid and thyroid hormone receptors, andactivating said receptor.
 5. A modified form of a co-suppressor having astructure and function characteristic of the silencing mediator forretinoic acid and thyroid hormone receptors selected from the groupconsisting of: full length silencing mediator for retinoic acid andthyroid hormone receptors plus GAL4 DNA binding domain, full lengthsilencing mediator for retinoic acid and thyroid hormone receptors plusGALA activation domain, and full length silencing mediator for retinoicacid and thyroid hormone receptors plus glutathione S-transferase (GST)tag.
 6. A modified co-suppressor according to claim 5, wherein saidmodified co-suppressor comprises full length silencing mediator forretinoic acid and thyroid receptors plus GAL4 activation domain.
 7. Amethod to identify compounds which disrupt the ability of aco-suppressor having a structure and function characteristic of thesilencing mediator for retinoic acid and thyroid receptors to complexwith steroid/thyroid hormone receptors, without substantially activatingsaid receptor, said method comprising: comparing the reporter signalproduced by two different expression systems in the absence and presenceof test compound, wherein said first expression system comprises acomplex comprising: a modified co-suppressor according to claim 6, ahomodimeric or heterodimeric member of the steroid/thyroid hormonesuperfamily of receptors selected from thyroid hormone receptorhomodimer, thyroid hormone receptor-retinoid X receptor heterodimer,retinoic acid receptor homodimer or retinoic acid receptor-retinoid Xreceptor heterodimer, and a response element for said member of thesteroid/thyroid hormone superfamily of receptors, wherein said responseelement is operatively linked to a reporter, and wherein said secondexpression system comprises a complex comprising: said modifiedco-suppressor, a homodimeric or heterodimeric form of the same member ofthe steroid/thyroid hormone superfamily of receptors as employed in saidfirst expression system, wherein said member is mutated such that itretains hormone dependent activation activity but has lost its abilityto repress basal level promoter activity of target genes, and the sameresponse element-reporter combination as employed in said firstexpression system, and thereafter selecting those compounds whichprovide: a lower reporter signal upon exposure of said compound to saidfirst expression system, relative to reporter signal in the absence ofsaid compound, and substantially the same reporter signal upon exposureof said compound to said second expression system, relative to reportersignal in the absence of said compound, wherein said selected compoundsare capable of disrupting the ability of a co-suppressor having astructure and function characteristic of the silencing mediator forretinoic acid and thyroid receptors to complex with steroid/thyroidhormone receptors, without substantially activating said receptor.
 8. Amethod according to claim 7 wherein said mutant receptor is selectedfrom RAR403 homodimers, RAR403-containing heterodimers, TR160 homodimersor TR160-containing heterodimers.
 9. A method according to claim 7,wherein the host is a mammalian or yeast cell.
 10. A method to identifycompounds which activate steroid/thyroid hormone receptor activity, butsubstantially lack the ability to disrupt a complex comprising asteroid/thyroid hormone receptor and a co-suppressor having a structureand function characteristic of the silencing mediator for retinoic acidand thyroid receptors, said method comprising: comparing the reportersignal produced by two different expression systems in the absence andpresence of test compound, wherein said first expression systemcomprises a complex comprising: a modified co-suppressor according toclaim 6, a homodimeric or heterodimeric member of the steroid/thyroidhormone superfamily of receptors selected from thyroid hormone receptorhomodimer, thyroid hormone receptor-retinoid X receptor heterodimer,retinoic acid receptor homodimer or retinoic acid receptor-retinoid Xreceptor heterodimer, and a response element for said member of thesteroid/thyroid hormone superfamily of receptors, wherein said responseelement is operatively linked to a reporter, and wherein said secondexpression system comprises: said modified co-suppressor, a homodimericor heterodimeric form of the same member of the steroid/thyroid hormonesuperfamily of receptors as employed in said first expression system,wherein said member is mutated such that it retains hormone dependentactivation activity but has lost its ability to repress basal levelpromoter activity of target genes, and the same responseelement-reporter combination as employed in said first expressionsystem, and thereafter selecting those compounds which provide: a higherreporter signal upon exposure of said compound to said second expressionsystem, relative to reporter signal in the absence of compound, andsubstantially the same reporter signal upon exposure of said compound tosaid first expression system, relative to reporter signal in the absenceof compound, wherein said selected compounds are capable of activatingsteroid/thyroid hormone receptor activity, but substantially lack theability to disrupt the complex of a co-suppressor having a structure andfunction characteristic of the silencing mediator for retinoic acid andthyroid receptors and a steroid/thyroid hormone receptor.
 11. A methodaccording to claim 10, wherein the host is a mammalian or yeast cell.12. A method to identify compounds which activate a steroid/thyroidhormone receptor, and disrupt the ability of a co-suppressor having astructure and function characteristic of the silencing mediator forretinoic acid and thyroid receptors to complex with said receptor, saidmethod comprising: comparing the reporter signal produced by twodifferent expression systems in the absence and presence of testcompound, wherein said first expression system comprises a complexcomprising: a modified co-suppressor according to claim 6, a homodimericor heterodimeric member of the steroid/thyroid hormone superfamily ofreceptors selected from thyroid hormone receptor homodimer, thyroidhormone receptor-retinoid X receptor heterodimer, retinoic acid receptorhomodimer or retinoic acid receptor-retinoid X receptor heterodimer, anda response element for said member of the steroid/thyroid hormonesuperfamily of receptors, wherein said response element is operativelylinked to a reporter, and wherein said second expression systemcomprises a complex comprising: said modified co-suppressor, the samehomodimeric or heterodimeric member of the steroid/thyroid hormonesuperfamily of receptors as employed in said first expression system,wherein said member is mutated such that it retains hormone dependentactivation activity but has lost its ability to repress basal levelpromoter activity of target genes, and the same responseelement-reporter combination as employed in said first expressionsystem, and thereafter selecting those compounds which provide: areduction in reporter signal upon exposure of compound to said firstexpression system, relative to reporter signal in the absence of saidcompound, and increased reporter signal upon exposure of compound tosaid second expression system, relative to reporter signal in theabsence of said compound, wherein said selected compounds are capable ofactivating a steroid/thyroid hormone receptor and disrupting a complexcomprising steroid/thyroid hormone receptor and a co-suppressor having astructure and function characteristic of the silencing mediator forretinoic acid and thyroid receptors.
 13. A method according to claim 12,wherein the host is a mammalian or yeast cell.
 14. A method to identifycompounds which activate a steroid/thyroid hormone receptor and/ordisrupt the ability of a co-suppressor having a structure and functioncharacteristic of the silencing mediator for retinoic acid and thyroidreceptors to complex with said receptor, said method comprising:comparing the reporter signals produced by a combination expressionsystem in the absence and presence of test compound, wherein saidcombination expression system comprises: a first homodimeric orheterodimeric member of the steroid/thyroid hormone superfamily ofreceptors selected from thyroid hormone receptor homodimer, thyroidhormone receptor-retinoid X receptor heterodimer, retinoic acid receptorhomodimer, or retinoic acid receptor-retinoid X receptor heterodimer, asecond homodimeric or heterodimeric form of the same member of thesteroid/thyroid hormone superfamily of receptors as employed in saidfirst homodimer or heterodimer, wherein said member is mutated such thatit retains hormone dependent activation activity but has lost itsability to repress basal level promoter activity of target genes,wherein either said first homodimer (or heterodimer) or said secondhomodimer (or heterodimer) is operatively linked to a GAL4 DNA bindingdomain, a response element for said member of the steroid/thyroidhormone superfamily of receptors, wherein said response element isoperatively linked to a first reporter, a GAL4 response element, whereinsaid response element is operatively linked to a second reporter, andthereafter identifying as capable of relieving the suppression ofsteroid/thyroid hormone receptor activity caused by a co-suppressorhaving a structure and function characteristic of the silencing mediatorfor retinoic acid and thyroid receptors, but substantially lacking theability to activate steroid/thyroid hormone receptor activity thosecompounds which provide: a higher reporter signal from the reporterresponsive to the first member upon exposure of said compound to saidfirst member, relative to reporter signal in the absence of saidcompound, and substantially the same reporter signal from the reporterresponsive to the second member upon exposure of said compound to saidsecond member, relative to reporter signal in the absence of saidcompound, or identifying as capable of activating steroid/thyroidhormone receptor activity, but substantially lacking the ability torelieve the suppression caused by a co-suppressor having a structure andfunction characteristic of the silencing mediator for retinoic acid andthyroid receptors those compounds which provide: a higher reportersignal from the reporter responsive to the second member upon exposureof said compound to said second member, relative to reporter signal inthe absence of compound, and substantially the same reporter signal fromthe reporter responsive to the first member upon exposure of saidcompound to said first member, relative to reporter signal in theabsence of said compound, or identifying as capable of relieving thesuppression of steroid/thyroid hormone receptor activity caused by aco-suppressor having a structure and function characteristic of thesilencing mediator for retinoic acid and thyroid receptors, andactivating said receptor those compounds which provide: increasedreporter signal from the reporter responsive to the second member uponexposure of said compound to said second member, relative to reportersignal in the absence of said compound, and substantially increasedreporter signal from the reporter responsive to the first member uponexposure of said compound to said first member, relative to reportersignal in the absence of said compound.
 15. A method according to claim14 wherein said combination expression system further comprises aco-suppressor of steroid/thyroid hormone receptor activity, saidco-suppressor having a structure and function characteristic of thesilencing mediator for retinoic acid and thyroid receptors.
 16. A methodto identify compounds which activate a steroid/thyroid hormone receptorand/or disrupt the ability of a co-suppressor having a structure andfunction characteristic of the silencing mediator for retinoic acid andthyroid receptors to complex with said receptor, said method comprising:comparing the reporter signals produced by a combination expressionsystem in the absence and presence of test compound, wherein saidcombination expression system comprises: a modified co-suppressoraccording to claim 6, a first homodimeric or heterodimeric member of thesteroid/thyroid hormone superfamily of receptors selected from thyroidhormone receptor homodimer, thyroid hormone receptor-retinoid X receptorheterodimer, retinoic acid receptor homodimer, or retinoic acidreceptor-retinoid X receptor heterodimer, a second homodimeric orheterodimeric form of the same member of the steroid/thyroid hormonesuperfamily of receptors as employed in said first homodimer orheterodimer, wherein said member is mutated such that it retains hormonedependent activation activity but has lost its ability to repress basallevel promoter activity of target genes, wherein either said firsthomodimer (or heterodimer) or said second homodimer (or heterodimer) isoperatively linked to a GALA DNA binding domain, a response element forsaid member of the steroid/thyroid hormone superfamily of receptors,wherein said response element is operatively linked to a first reporter,a GAL4 response element, wherein said response element is operativelylinked to a second reporter, and thereafter identifying as capable ofdisrupting the ability of a co-suppressor having a structure andfunction characteristic of the silencing mediator for retinoic acid andthyroid receptors to complex with steroid/thyroid hormone receptor,without substantially activating steroid/thyroid hormone receptor, thosecompounds which provide: a lower reporter signal from the reporterresponsive to the first member upon exposure of said compound to saidfirst member, relative to reporter signal in the absence of saidcompound, and substantially the same reporter signal from the reporterresponsive to the second member upon exposure of said compound to saidsecond member, relative to reporter signal in the absence of saidcompound, or identifying as capable of activating steroid/thyroidhormone receptor activity, but substantially lacking the ability todisrupt a complex comprising a steroid/thyroid hormone receptor and aco-suppressor having a structure and function characteristic of thesilencing mediator for retinoic acid and thyroid receptors thosecompounds which provide: a higher reporter signal from the reporterresponsive to the second member upon exposure of said compound to saidsecond member, relative to reporter signal in the absence of compound,and substantially the same reporter signal from the reporter responsiveto the first member upon exposure of said compound to said first member,relative to reporter signal in the absence of said compound, oridentifying as capable of disrupting a complex comprising asteroid/thyroid hormone receptor and a co-suppressor having a structureand function characteristic of the silencing mediator for retinoic acidand thyroid receptors, and activating said receptor those compoundswhich provide: a reduction in reporter signal from the reporterresponsive to the first member upon exposure of said compound to saidfirst member, relative to reporter signal in the absence of saidcompound, and increased reporter signal from the reporter responsive tothe second member upon exposure of said compound to said second member,relative to reporter signal in the absence of said compound.
 17. Amethod to identify compounds which modulate the interaction of ahomodimeric or heterodimeric member of the steroid/thyroid hormonesuperfamily of receptors with a co-suppressor having a structure andfunction characteristic of the silencing mediator for retinoic acid andthyroid hormone receptors, said method comprising determining the effectof a test compound on the formation or disruption of a complex whichcomprises said co-suppressor and said member, wherein said membercontains a silencing domain which represses basal level promoteractivity of target genes.
 18. A method according to claim 17, whereinsaid complex further comprises a response element for said member of thesteroid/thyroid hormone superfamily of receptors.
 19. A method toidentify compounds which modulate the interaction of a homodimeric orheterodimeric member of the steroid/thyroid hormone superfamily ofreceptors with a co-suppressor having a structure and functioncharacteristic of the silencing mediator for retinoic acid and thyroidhormone receptors, said method comprising determining the effect of atest compound on the level of reporter signal produced by an expressionsystem comprising (i) said member, wherein said member contains asilencing domain which represses basal level promoter activity of targetgenes, (ii) said co-suppressor, and (iii) a response element for saidmember operatively linked to a reporter gene, wherein a change in thereporter signal in the presence of said test compound is indicative of acompound that modulates said interaction.
 20. A method according toclaim 19, wherein the host is a mammalian or yeast cell.
 21. A method toidentify compounds which modulate the interaction of a homodimeric orheterodimeric member of the steroid/thyroid hormone superfamily ofreceptors with a co-suppressor having a structure and functioncharacteristic of the silencing mediator for retinoic acid and thyroidhormone receptors, said method comprising determining the effect of atest compound on the level of reporter signal produced by an expressionsystem comprising (i) a modified member of the steroid/thyroid hormonesuperfamily of receptors, wherein said modified member contains anactivation domain which renders said receptor constitutively active,(ii) a fusion protein comprising the receptor interaction domain of saidco-suppressor operatively linked to the GAL4 DNA binding domain, and(iii) a GAL4 response element operatively linked to a reporter gene,wherein a change in the reporter signal in the presence of said testcompound is indicative of a compound that modulates said interaction.22. A method to dissociate a complex comprising a co-suppressor ofsteroid/thyroid hormone receptor activity, said co-suppressor having astructure and function characteristic of the silencing mediator forretinoic acid and thyroid hormone receptors and a homodimeric orheterodimeric member of the steroid/thyroid hormone superfamily ofreceptors, wherein said member contains a silencing domain whichrepresses basal level promoter activity of target genes, said methodcomprising contacting said complex with ligand for said member of thesteroid/thyroid hormone superfamily of receptors.
 23. A method torepress the activity of a member of the steroid/thyroid hormonesuperfamily of receptors containing a silencing domain which repressesbasal level promoter activity of target genes, said method comprisingcontacting said member of the steroid/thyroid hormone superfamily ofreceptors with a sufficient quantity of a co-suppressor having astructure and function characteristic of the silencing mediator forretinoic acid and thyroid receptors so as to repress the activity ofsaid member.
 24. A method according to claim 23 wherein said member ofthe steroid/thyroid hormone superfamily of receptors is selected fromthyroid hormone receptor, retinoic acid receptor, vitamin D receptor orperoxisome proliferator activated receptor.
 25. An antibody raisedagainst a co-suppressor of steroid/thyroid hormone receptor activity,said co-suppressor having a structure and function characteristic of thesilencing mediator for retinoic acid and thyroid hormone receptors. 26.A method to block the repressing effect of co-suppressors having astructure and function characteristic of the silencing mediator forretinoic acid and thyroid receptors, said method comprisingadministering an effective amount of an antibody according to claim 25.